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© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• 1970s: Gene cloning
– Clone
– Restriction Enzymes
1
9/24/2018
Key Concepts
• Restriction Enzymes
–bacteria
–phosphodiester bond
–Bind to, recognize, and cut DNA
–restriction site
• same forward and backward
–4 or 6 bp cutters
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
2
9/24/2018
Key Concepts
• Restriction enzymes
b."blunt" ends
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
3
9/24/2018
Key Concepts
• Plasmid DNA
• extrachromosomal DNA
• approximately 1 to 4 kb
• replicate independently of
chromosome
• vectors
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Creating recombinant DNA
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
4
9/24/2018
Key Concepts
• Transformation of Bacterial Cells
–electroporation
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Selection of recombinant bacteria after transformation
-Antibiotic selection
-Blue-white selection
artificial lactose
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9/24/2018
Key Concepts
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
6
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• human gene cloning
insulin
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
7
9/24/2018
Key Concepts
• Types of Vectors
– Bacterial plasmid vectors
inserts that are smaller than 7 kb
– Bacteriophage vectors
– Cosmid vectors
– Expression vectors
– Bacterial Artificial Chromosomes (BAC)
– Yeast Artificial Chromosomes (YAC)
– Ti vectors
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Bacteriophage vectors
– advantage: clone up to 25 kb
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
8
9/24/2018
Key Concepts
• Cosmid vectors:
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Expression vectors
– Bacteria expression vector:
• prokaryotic promoter site
• Bacterial RNA POL can bind to promoter
• Problems?
-bacteria ribosomes cannot translate
eukaryote sequence
-protein is not folded correctly
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
9
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
10
9/24/2018
Key Concepts
• Bacteria artificial chromosomes
(BAC)
–Large low copy plasmids
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Yeast artificial chromosomes (YAC)
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
11
9/24/2018
Key Concepts
• Ti vector
–isolated from the bacterium
–bacteria infects plant cells
–T DNA from the Ti plasmid inserts
into the host chromosome
–T DNA codes for auxin
–use Ti vectors to deliver genes to
plants
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
12
9/24/2018
Key Concepts
• Creating DNA Libraries
Key Concepts
–Disadvantages of genomic
libraries
•Introns are cloned in addition to
exons
•very large genome
•Time consuming!
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
13
9/24/2018
Key Concepts
• cDNA Libraries
– Advantage over genomic libraries
• Collection of actively expressed genes
• Introns are NOT cloned
• isolate genes that are primarily expressed
only under certain conditions
– Disadvantage
• source tissue with an abundant amount of
mRNA
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Colony hybridization
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
14
9/24/2018
Key Concepts
• Polymerase Chain Reaction
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
15
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
16
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
17
9/24/2018
Key Concepts
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
–Disadvantage
• Need to know something about the
DNA sequence that flanks the gene
of interest to design primers
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
18
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
19
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• Agarose Gel Electrophoresis
–buffer solution
–wells at the top of the gel
–Electric current is applied through
electrodes at opposite ends of the
gel
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
20
9/24/2018
Key Concepts
• Agarose Gel Electrophoresis
-Migration distance
–Tracking dye
–DNA staining dyes
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
• Here is a result in which you ran your DNA fragments on a 1% gel. What
is the approximate size of band B? Why did it not migrate super fast
through the gel?
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
21
9/24/2018
• Here is a result in which you ran your DNA fragments on a 1% gel. What
is the approximate size of band B? Why did it not migrate super fast
through the gel? B is 1250bp; B is not the smallest fragment
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• DNA Sequencing
–Sanger
–Roche 454
–ABI SOLID
–Nanotechnology
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
22
9/24/2018
Key Concepts
• Fluorescence in situ
hybridization (FISH)
-Chromosome Location
-Copy Number
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
23
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
–Southern blotting
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
24
9/24/2018
Key Concepts
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
Microarrays
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
25
9/24/2018
Key Concepts
Gene microarrays
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
RNA
interference
(RNAi)
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
26
9/24/2018
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
27
9/24/2018
Key Concepts
• Bioinformatics: Merging molecular
biology with computer technology
• Application
–Databases
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
–GenBank
• accession number
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9/24/2018
Key Concepts
• Basic Local Alignment Search
Tool (BLAST)
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Key Concepts
• The Human Genome Project
–Started in 1990 by the U.S. Department
of Energy
–International collaborative effort
–China, France, Germany, Great Britain,
Japan, and the United States
–Competitor: Celera Genomics
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
29
9/24/2018
Key Concepts
• The Human Genome Project
– April 14, 2003: map of the human genome was completed
– Consists of 20,000 protein-coding genes
– Map was complete with virtually all bases identified and
placed in their order and potential genes assigned to
chromosomes
• Why are there only 20,000 genes coding for proteins when it
was predicted that there would be 100,000 genes?
© 2013 Pearson Education, Inc. Thieman, W.J. and Palladino, M.A. Introduction to Biotechnology. 2012. Pearson. PowerPoint Lecture by
Melissa Rowland-Goldsmith, Chapman University; modified from the aforementioned PowerPoint Lecture
Next Class:
BIO374 Modern Biotechnology
Proteins as Products
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